Project Summary Sleep disturbances are increasingly common and are associated with a variety of comorbidities and other public health consequences. It is therefore critical to improve our understanding of the neural mechanisms that control the timing and quality of sleep. Key signaling molecules that regulate sleep in animals ranging from flies to humans come from the family of neuropeptide transmitters. These molecules have sparse expression levels and selective effects on behavior, including sleep, making them prime candidates for the development of focused drug treatments with minimal side effects. However, the mechanisms by which these molecules act individually and in concert to regulate target cells in the brain and thus behavior are poorly understood. This proposal will take advantage of the powerful genetics and relatively simple sleep network organization of the fruit fly, Drosophila melanogaster, to address how neuropeptides function at the molecular, cellular, and behavioral levels to regulate sleep. In Aim 1, we will perform live fluorescent imaging to identify cellular targets and intracellular signaling mechanisms downstream of the receptor for the newly described sleep regulator, neuropeptide F (NPF), and determine how this molecule interacts with two additional established sleep regulators, short neuropeptide F (sNPF) and pigment dispersing factor (PDF). In Aim 2, we will carry out optogenetic activation of neuronal populations producing these same neuropeptides either individually or in combination, and record the effects on sleep behavior. In combination, the proposed work will allow us to establish the causal roles of key neuropeptide signaling molecules in the control of sleep, expanding our understanding of the mechanisms that control sleep and wakefulness at the molecular and circuit levels. In turn, this knowledge will provide a basis for the design of more effective treatments of human sleep abnormalities. This R15 AREA proposal will directly involve undergraduate students in all aspects of the research, including designing experiments, carrying out studies involving techniques of genetic manipulation, molecular and cellular neurobiology, and behavioral analysis. Their experiences will provide formative training for future careers in biomedical fields.